Petasis Sequence Reactions for the Scaffold-Diverse Synthesis of Bioactive Polycyclic Small Molecules

The multicomponent Petasis reaction is a versatile method to access functionalized amines. The combination of Petasis reaction with subsequent ring-closing reactions is a powerful strategy to build novel polycyclic scaffolds. In this study, we report the generation of a diverse set of small molecules with polycyclic scaffolds featuring a high content of sp3-hybridized carbon atoms and multiple stereogenic centers by employing three-component Petasis reaction (3C-PR)—Intramolecular Diels–Alder (IMDA) and 3C-PR—ring-closing metathesis (RCM)—IMDA sequence reactions. This work demonstrates the wide substrate tolerance and broad applicability to access unexplored polycyclic scaffolds of biological interest using Petasis sequence reactions.


■ INTRODUCTION
Small molecules are valuable therapeutics for treating human diseases and useful tools in probing biological functions of proteins, human physiology, and numerous cellular activities. One limitation in the current field of small-molecule research lies in the limited coverage of "chemical space" in synthetic molecules and commercially available screening libraries, which represent merely a tiny fraction of all possible structural complexity. The vastness of unexplored chemical space spurred chemists to develop efficient approaches to populate the current reservoir of small molecules and compound libraries with compounds covering bioactivity-related chemical space. 1−3 One such approach is to build compound collections with high structural diversity and coverage on unexplored chemical space. 4−9 A wide range of synthetic methods have been applied for the synthesis of sp 3 -rich polycyclic bioactive small molecules. 10−12 Recent synthetic examples include the solidphase synthesis of DNA-tagged heterocycles, 13 rhodiumcatalyzed intramolecular annulations, 14 and visible-light-photocatalyzed synthesis of N-heterospirocyles. 15 Compounds that are structurally complex are likely to interact with biomacromolecules with different selectivity and specificity profiles. 7,16 An efficient synthetic method to access compounds of diverse structures is the employment of multicomponent reactions (MCRs), which are highly relevant to medicinal chemistry and drug discovery as MCRs enable the construction of diverse heterocyclic scaffolds with a high degree of complexity and stereoselectivity. 17,18 Multicomponent Petasis reaction (PR) enables the preparation of highly functionalized amines from a primary or secondary amine, a boronic acid, and a carbonyl component. 19−22 PR products are amenable precursors for sequence reactions and are compatible with the combination of various secondary transformations to yield compounds with polycyclic scaffolds, such as PR�ring-closing metathesis (RCM) ( Figure 1A), PR�intramolecular Diels−Alder (IMDA) ( Figure 1B), and PR/IMDA in conjunction with other cyclization reactions ( Figure 1C and 1D). Examples reported in the past decade demonstrated the great value of PR in synthesizing structurally diverse small molecules, natural products, and molecular libraries. 22−32 In this context, we are continuing our efforts aiming to develop efficient methods for the construction of new polycyclic scaffolds for small molecule discovery. In this study, we report two synthetic sequences involving the multicomponent PR to form synthetically tractable small molecules of diverse structural complexity that are evaluated as bioactive compounds in early drug discovery stages (Figure 2).
Preliminary cyclization was performed based on the Petasis products 4e−4i that contained the olefin appendages for an IMDA reaction. We envisioned that two cyclic scaffolds, epoxyisoindole and epoxybenzoazepine, would be formed, while after trying under different cyclization conditions, only the formation of the epoxyisoindole scaffold was observed. The furan-2-ylmethyl substituted Petasis products 4f, 4h, and 4i gave the epoxyisoindoles 5a−5c in 50−59% yields (Scheme 2). The thiophen-2-ylmethyl substituted Petasis product 4g failed to cyclize under the refluxing condition even with a prolonged duration. All products were obtained as a single diastereomer. The exo-product of the IMDA reaction was confirmed by singlecrystal X-ray analysis of compound 5a.
Given the PR�IMDA results using allylated Petasis products, we continued the synthesis of propargylated Petasis products 7 with the aim of obtaining new polycyclic scaffolds via IMDA or RCM between the positioned allyl-and propargyl substituents. N-propargyl-thiophenemethylamine 6 was synthesized by reported procedures and used as the amine component. 34 Using 5-allyl-2,2-dimethyl-1,3-dioxolan-4-ol 2 and arylboronic acids, the corresponding Petasis products 7a−7d were obtained in yields up to 91% (Scheme 3). The subsequent IMDA reactions using 7a−7d as the substrate did not lead to any IMDA cyclized products under refluxing conditions. Therefore, we switched our efforts in screening different ruthenium catalysts to perform the enyne metathesis reaction to generate cyclized scaffolds.
The use of Grubbs second generation catalyst resulted in the dimerization of Petasis product 7 with only a trace amount of expected cyclized product 8 (Scheme 4). The use of Grubbs first generation catalyst afforded the cyclized products 8a−8d in improved yet poor yields, which were subjected to a further intermolecular Diels−Alder (DA) reaction in the presence of  activated dienophiles to form bicyclic compounds 9 with a pyridazino[4,3-c]azepine scaffold (Scheme 5). A selection of the activated dienophiles including azodicarboxylic dimorpholide and 1,1′-(azodicarbonyl)dipiperidine gave the desired products in yields ranging from 55−77% as diastereomeric mixtures with the endo-product being the major diastereomer. The major endoproduct was confirmed by single-crystal X-ray analysis of compounds 9a and 9j. The pyridazino[4,3-c]azepines 9 can be subject to further decoration, e.g., compounds 9e and 9f can be Boc-deprotected and functionalized on the resulting hydrazine amines.
To probe the medicinal chemistry properties, we calculated the molecular and absorption-distribution-metabolism-excretion (ADME) properties of synthesized compounds 5 and 9 (Tables S1 and S2), which revealed that many compounds showed favorable predicted drug-likeness properties. The biological relevance of the obtained pyridazino[4,3-c]azepines 9 was demonstrated by an antiproliferation assay, in which the tested compounds exhibited 40−90% inhibition against human acute myeloid leukemia cells MOLM-13 and the human choriocarcinoma cells JAR at a concentration of 10 μM. Compounds that showed at least 50% inhibition at 10 μM were further tested for their IC 50 values, which revealed single-digit micromolar potency for selected compounds 9a, 9b, 9e, and 9f (e.g., azepine 9e, IC 50 : 5.3 μM against MOLM-3 and 9.0 μM against JAR cells, respectively, Figure 3).

■ CONCLUSION
We employed 3C-PR�IMDA and 3C-PR�RCM�IMDA sequences of robust and complexity-generating reactions to yield new small molecules with epoxyisoindole and pyridazinoazepine scaffolds. The sequence reactions were performed in straightforward and mild conditions with easily accessible substrates. The Petasis products and generated polycyclic compounds feature a high content of sp 3 -hybridized atoms and are amenable to be appended with other functional groups. The biological relevance of the obtained compounds has been tested in the antiproliferation assay against human cancer cell lines. The work demonstrated the broad applicability to access unexplored polycyclic scaffolds of biological interest using 3C-PR-involved sequence reactions. ■ EXPERIMENTAL SECTION General Information. All commercially available solvents and reagents were purchased from Sigma-Aldrich, TCI Chemical, or Fisher Scientific and used without further purification. All reactions were monitored by thin layer chromatography (TLC) and an LC-MS Agilent 1260 II Infinity system equipped with a mass detector (column: InfinityLab Poroshell 120 EC-C18, 2.1 × 150, 2.7 μm). Appropriate gradient systems were applied by mixing H 2 O (+ 0.1% TFA) and acetonitrile (+ 0.1% TFA). Analytical thin-layer chromatography was carried out using Merck silica gel aluminum plates with F-254 indicator, visualized under UV light (at 254 nm), iodine stain, or dipping in potassium permanganate stain (1.5 g of KMnO 4 , 10 g of K 2 CO 3 , 1.25 mL of 10% aqueous NaOH solution, and 200 mL of water). Starting materials 1c, 2, 6a, and 6b were prepared following the known literature procedures and the characterization data were in agreement with the literature data. The products were purified by column chromatography over silica gel (Merck 60 particle size 0.040−0.063 mm). Solvents for chromatography were laboratory grade. All 1 H and 13 C NMR spectra were recorded on a Bruker DRX400 (400 MHz), DRX500 (500 MHz), DRX600 (600 MHz), and DRX700 (700 MHz) spectrometers in CDCl 3 and (CD 3 ) 2 SO. Data are reported in the following order: chemical shift in ppm; multiplicities are indicated s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet), ddt (doublet of doublet of triplets), and m (multiplet). Coupling constants (J) are given in Hertz (Hz). High-resolution mass spectra were recorded on an LTQ Orbitrap mass spectrometer coupled to an Accela HPLC System (HPLC column: Hypersyl GOLD, 50 mm × 1 mm, 1.9 μm). Chemical yields refer to isolated pure substances.
General Procedure for the Synthesis of Allylated Tertiary Amines 4a−4i.
PR/RCM product 8b (70 mg, 0.22 mmol, 1 equiv) was dissolved in toluene (0.5 M) and then azodicarboxylic dimorpholide (62.15, 0.24 mmol, 1.1 equiv) was added. The reaction mixture was stirred under the reflux condition overnight. The solvent was evaporated and the product was purified as an off-white powder in 73% yield (dr 2:1) by column chromatography using 70% ethyl acetate in petroleum ether (isocratic flow). The major diastereomer: 1